Erythropoietin (hereinafter EPO) is a circulating glycoprotein, which stimulates erythrocyte formation in higher organisms. See, Carnot et al., Compt. Rend., 143:384 (1906). As such, EPO is sometimes referred to as an erythropoiesis stimulating factor.
The life of human erythrocytes is about 120 days. Thus, about 1/120 of the total erythrocytes are destroyed daily in the reticulo-endothelial system. Concurrently, a relatively constant number of erythrocytes are produced daily to maintain the level of erythrocytes at all times (Guyton, Textbook of Medical Physiology, pp. 56-60, W. B. Saunders Co., Philadelphia (1976)).
Erythrocytes are produced by the maturation and differentiation of the erythroblasts in bone marrow, and EPO is a factor which acts on less differentiated cells and induces their differentiation to erythrocytes (Guyton, supra).
EPO is a promising therapeutic agent for the clinical treatment of anemia or, in particular, renal anemia. Unfortunately, the use of EPO is not yet common in practical therapy due to its low availability.
For EPO to be used as a therapeutic agent, consideration should be given to possible antigenicity problems, and it is therefore preferable that EPO be prepared from a raw material of human origin. For example, human blood or urine from patients suffering from aplastic anemia or like diseases who excrete large amounts of EPO may be employed. These raw materials however, are in limited supply. See, for example, White et al., Rec. Progr. Horm. Res., 16:219 (1960); Espada et al., Biochem. Med., 3:475 (1970); Fisher, Pharmacol. Rev., 24:459 (1972) and Gordon, Vitam. Horm. (N.Y.) 31:105 (1973), the disclosures of which are incorporated herein by reference.
The preparation of EPO products has generally been via the concentration and purification of urine from patients exhibiting high EPO levels, such as those suffering from aplastic anemia and like diseases. See for example, U.S. Pat. Nos. 4,397,840, 4,303,650 and 3,865,801 the disclosures of which are incorporated herein by reference. The limited supply of such urine is an obstacle to the practical use of EPO, and thus it is highly desirable to prepare EPO products from the urine of healthy humans. A problem in the use of urine from healthy humans is the low content of EPO therein in comparison with that from anemic patients. In addition, the urine of healthy individuals contains certain inhibiting factors which act against erthropoiesis in sufficiently high concentration so that a satisfactory therapeutic effect would be obtained from EPO derived therefrom only following significant purification.
EPO can also be recovered from sheep blood plasma, and the separation of EPO from such blood plasma has provided satisfactorily potent and stable water-soluble preparations. See, Goldwasser, Control Cellular Dif. Develop., Part A; pp 487-494, Alan R. Liss, Inc., N.Y., (1981), which is incorporated herein by reference. Sheep EPO would, however, be expected to be antigenic in humans.
Thus, while EPO is a desirable therapeutic agent, conventional isolation and purification techniques, used which natural supply sources, are inadequate for the mass production of this compound.
Sugimoto et al., in U.S. Pat. No. 4,377,513 describe one method for the mass production of EPO comprising the in vivo multiplications of human lymphoblastoid cells, including Namalwa, BALL-1, NALL-1, TALL-1 and JBL.
For therapeutic use of EPO, it is necessary to purify the EPO protein to homogeneity.